2.2 Site-specific PEGylation of FGF2 conjugates and
purification
The PEGylated FGF2 conjugates were generated by site specific PEGylation
using mPEG-Maleimide (mPEG-MAL, 5kDa). We optimized the reaction
conditions, including temperature, reaction time, and the PEG-to-FGF2
molar ratio as 4 °C a PEG-to-FGF2 molar ratio of 4, and 1 h,
respectively. The yield of PEGylation was confirmed by SDS-PAGE.
To separate the PEGylated FGF2 conjugates from unreacted FGF2 and
mPEG-MAL, reaction mixtures were applied at a rate of 5 mL/min to a
Heparin HP column (5 mL bed volume) pre-equilibrated with 15 column
volumes (CVs) of binding buffer (25 mM HEPES buffer, pH 7.5). The column
was then washed with 10 CVs of binding buffer, and then the protein was
eluted with buffer A (25 mM HEPES buffer, pH 7.5 and buffer B (25 mM
HEPES buffer, pH 7.5, 2 M NaCl) over 30 CVs. Eluted fractions were
collected and analyzed by SDS-PAGE.